PROJECT SUMMARY The cannabinoid receptor (CB1) is the most abundantly expressed GPCR in the central nervous system, and the target of drugs of abuse like the psychoactive components in marijuana and synthetic cannabinoids. While CB1 is also a potentially high-value therapeutic target, exploiting this key receptor for therapeutic goals has been complicated by a wide array of undesired side-effects. One explanation for these side-effects has implicated the promiscuous nature of CB1 when activated, as CB1 is capable of coupling with a variety of signaling partners. Canonically, CB1 couples to inhibitory G-protein subtypes (G?i/o). However, some CB1 ligands also cause the receptor to couple with other G-protein subtypes, such as G?s and G?q. Thus, some compounds targeting CB1 can exhibit ligand bias?a phenomenon whereby ligand binding to a receptor stabilizes a unique receptor conformation that selectively promotes (or inhibits) interactions with different signaling partners. CB1 signaling can also be modulated by ligands that bind allosterically, outside the normal (orthosteric) ligand binding pocket. One CB1 allosteric ligand, ORG27569 (ORG), shows especially peculiar behavior?it increases binding of agonists binding to CB1, yet inhibits receptor activation of G-proteins. While investigating the structural mechanisms underlying this apparent paradox, our lab recently found that ORG-binding stabilizes a unique CB1 conformation, one in which the conformational changes necessary for G-protein coupling are restricted. While this unique conformation has been shown to reduce Gi-mediated signaling, its full physiological role remains controversial. Moreover, how and why ORG increases agonist binding, and whether or not it causes other structural changes in the receptor is still not known. This proposal will explore these issues through three Specific Aims (SAs) designed to explore and define the molecular mechanisms involved in manipulation of CB1 by biased ligands and allosteric modulators. SA1 will define how ORG affects conformational changes that occur around the orthosteric ligand binding pocket in response to agonist binding, using novel fluorescence techniques. These experiments will determine if ORG induces alternate structures in this area, or if changes in this key region are decoupled from conformational changes in the signaling cytoplasmic domain. SA2 will directly test if the effect of allosteric ligands on CB1 require higher-order receptor multimers by carrying out fluorescent and radioligand binding studies of monomeric CB1 isolated in nanodsics. Finally, SA3 will develop and use novel biosensors to quantify and directly compare biochemical and pharmacological parameters underlying ligand bias and G-protein subtype selectivity at the CB1/G-protein/ligand signaling complex. Not only will these experiments address key questions about CB1, they will also provide vital experience for the trainee in both classical and cutting-edge methods in pharmacology, biochemistry, and biophysics used for the study of GPCR structure and function.